IL-13 protects from Clostridioides difficile colitis.

OBJECTIVES: Clostridioides difficile infection (CDI) is the leading hospital-acquired infection in North America. We have previously discovered that antibiotic disruption of the gut microbiota decreases intestinal IL-33 and IL-25 and increases susceptibility to CDI. We further found that IL-33 promotes protection through type 2 Innate Lymphoid Cells (ILC2s), which produce IL-13. However, the contribution of IL-13 to disease has never been explored. METHODS: We used a validated model of CDI in mice, in which we neutralized via blocking antibodies, or administered recombinant protein, IL-13 to assess the role of this cytokine during infection using weight and clinical scores. Fluorescent activated cell sorting (FACS) was used to characterize myeloid cell population changes in response to IL-13 manipulation. RESULTS: We found that administration of IL-13 protected, and anti-IL-13 exacerbated CDI. Additionally, we observed alterations to the monocyte/macrophage cells following neutralization of IL-13 as early as day three post infection. We also observed elevated accumulation of myeloid cells by day four post-infection following IL-13 neutralization. Neutralization of the decoy receptor, IL-13Rα2, resulted in protection from disease, likely through increased available endogenous IL-13. CONCLUSIONS: Our data highlight the protective role of IL-13 in protecting from more severe CDI and the association of poor responses with a dysregulated monocyte-macrophage compartment. These results increase our understanding of type 2 immunity in CDI and may have implications for treating disease in patients.

Fecal Microbiota Transplantation Increases Colonic IL-25 and Dampens Tissue Inflammation in Patients with Recurrent Clostridioides difficile.

Clostridioides difficile infection (CDI) is the most common hospital-acquired infection in the United States. Antibiotic-induced dysbiosis is the primary cause of susceptibility, and fecal microbiota transplantation (FMT) has emerged as an effective therapy for recurrence. We previously demonstrated in the mouse model of CDI that antibiotic-induced dysbiosis reduced colonic expression of interleukin 25 (IL-25) and that FMT protected in part by restoring IL-25 signaling. Here, we conducted a prospective study in humans to test if FMT induced IL-25 expression in the colons of patients with recurrent CDI (rCDI). Colonic biopsy specimens and blood were collected at the time of FMT and 60 days later. Colon biopsy specimens were analyzed for IL-25 protein levels, total tissue transcriptome, and epithelium-associated microbiota before and after FMT, and peripheral immune cells were immunophenotyped. FMT increased alpha diversity of the colonic microbiota and levels of IL-25 in colonic tissue. In addition, FMT increased expression of homeostatic genes and repressed inflammatory genes. Finally, circulating Th17 cells were decreased post-FMT. The increase in levels of the cytokine IL-25 accompanied by decreased inflammation is consistent with FMT acting in part to protect from recurrent CDI via restoration of commensal activation of type 2 immunity. IMPORTANCE Fecal microbiota transplantation (FMT) is an effective treatment for C. difficile infection for most patients; however, introducing a complex mixture of microbes also has had unintended consequences for some patients. Attempts to create a standardized probiotic therapeutic that recapitulates the efficacy of FMT have been unsuccessful to date. We sought to understand what immune markers are changed in patients undergoing FMT to treat recurrent C. difficile infection and identified an immune signaling molecule, IL-25, that was restored by FMT. This finding indicates that adjunctive therapy with IL-25 could be useful in treating C. difficile infection.

Vitamin-D status is not a confounder of the relationship between zinc and diarrhoea: a study in 6-24-month-old underweight and normal-weight children of urban Bangladesh.

BACKGROUND/OBJECTIVE: The role of micronutrients particularly zinc in childhood diarrhoea is well established. Immunomodulatory functions of vitamin-D in diarrhoea and its role in the effect of other micronutrients are not well understood. This study aimed to investigate whether vitamin-D directly associated or confounded the association between other micronutrient status and diarrhoeal incidence and severity in 6-24-month underweight and normal-weight children in urban Bangladesh. SUBJECTS/METHODS: Multivariable generalised estimating equations were used to estimate incidence rate ratios for incidence (Poisson) and severity (binomial) of diarrhoea on cohorts of 446 normal-weight and 466 underweight children. Outcomes of interest included incidence and severity of diarrhoea, measured daily during a follow-up period of 5 months. The exposure of interest was vitamin-D status at enrolment. RESULTS: Normal-weight and underweight children contributed 62 117 and 62 967 day observation, with 14.2 and 12.8 days/child/year of diarrhoea, respectively. None of the models showed significant associations of vitamin-D status with diarrhoeal morbidity. In the final model, zinc-insufficient normal-weight children had 1.3 times more days of diarrhoea than sufficient children (P<0.05). Again zinc insufficiency and mother's education (1-5 and >5 years) had 1.8 and 2.3 times more risk of severe diarrhoea. In underweight children, older age and female had 24-63 and 17% fewer days of diarrhoea and 52-54 and 31% fewer chances of severe diarrhoea. CONCLUSION: Vitamin-D status was not associated with incidence and severity of diarrhoea in study children. Role of zinc in diarrhoea was only evident in normal-weight children. Our findings demonstrate that vitamin-D is not a confounder of the relationship between zinc and diarrhoea.

Expression, purification, and evaluation of recombinant LecA as a candidate for an amebic colitis vaccine.

Entamoeba histolytica, which causes amebic colitis and liver abscess, is considered a major enteric pathogen in residents and travelers to developing countries where the disease is endemic. Interaction of this protozoan parasite with the intestine is mediated through the binding of the trophozoite stage to intestinal mucin and epithelium via a galactose and N-acetyl-d-galactosamine (Gal/GalNAc) lectin comprised of a disulfide linked heavy (ca. 180 kDa) and light chain (ca. 35 kDa) and a noncovalently bound intermediate subunit (ca. 150 kDa). Our efforts to develop a vaccine against this pathogen have focused on an internal 578 amino acid fragment, designated LecA, located within the cysteine-rich region of the heavy chain subunit because: (i) it is a major target of adherence-blocking antibodies of seropositive individuals and (ii) vaccination with his-tagged LecA provides protection in animal models. We developed a purification process for preparing highly purified non-tagged LecA using a codon-optimized gene expressed in Escherichia coli. The process consisted of: (i) cell lysis, collection and washing of inclusion bodies; (ii) solubilization and refolding of denatured LecA; and (iii) a polishing gel filtration step. The purified fragment existed primarily as a random coil with ß-sheet structure, contained low endotoxin and nucleic acid, was highly immunoreactive, and elicited antibodies that recognized native lectin and that inhibited in vitro adherence of trophozoites to CHO cells. Immunization of CBA mice with LecA resulted in significant protection against cecal colitis. Our procedure yields sufficient amounts of highly purified LecA for future studies on stability, immunogenicity, and protection with protein-adjuvant formulations.

Leptin and mucosal immunity.

Enhanced susceptibility to infection has long been recognized in children with congenital deficiency of leptin or its receptor. Studies in mice have demonstrated that leptin deficiency affects both the innate and acquired immune systems. Here, we review recent studies that demonstrate the impact on immunity of a common non-synonomous polymorphism of the leptin receptor. In a Bangladesh cohort of children, the presence of two copies of the ancestral Q223 allele was significantly associated with resistance to amebiasis. Children and mice with at least one copy of the leptin receptor 223R mutation were more susceptible to amebic colitis. Leptin signaling in the intestinal epithelium and downstream STAT3 (signal transducer and activator of transcription 3) and SHP2 (Src homology phosphatase 2) signaling were required for protection in the murine model of amebic colitis. Murine models have also implicated leptin in protection from other infections, including Mycobacterium tuberculosis, Klebsiella pneumoniae, and Streptococcus pneumoniae. Thus, the role of leptin signaling in infectious disease and specifically leptin-mediated protection of the intestinal epithelium will be the focus of this review.

Evidence for a link between locus R-R sequence type and outcome of infection with Entamoeba histolytica.

The results of Entamoeba histolytica infections range from asymptomatic colonization to variable disease outcomes. However, markers that may predict infection outcomes are not known. Here, we investigated sequence types of a non-coding tRNA-linked locus R-R to identify surrogate markers that may show association with infection outcomes. Among 112 clinical samples--21 asymptomatic, 20 diarrhoea/dysentery and 71 liver abscesses--we identified 11 sequence types. Sequence type 5RR was mostly associated with asymptomatic samples, and sequence type 10RR was predominantly associated with the symptomatic (diarrhoea/dysentery and liver abscess) samples. This is the first report that identifies markers that may predict disease outcomes in E. histolytica infection.

Leptin signaling in intestinal epithelium mediates resistance to enteric infection by Entamoeba histolytica.

Leptin is an adipocytokine that links nutrition to immunity. Previous observation that a genetic polymorphism in the leptin receptor affected susceptibility to Entamoeba histolytica infection led to the hypothesis that leptin signaling has a protective role during intestinal amebic infection. In this study we show that mice lacking the functional leptin receptor developed devastating mucosal destruction after E. histolytica infection. Bone marrow chimera experiments demonstrated that leptin receptor expressed on hematopoietic cells was not sufficient to confer resistance. Similarly, peripheral knockout of the leptin receptor rendered animals susceptible, indicating that central expression of the leptin receptor was not sufficient to confer protection. The site of leptin action was localized to the gut via an intestinal epithelium-specific deletion of the leptin receptor, which rendered mice susceptible to infection and mucosal destruction by the parasite. Mutation of tyrosine 985 or 1138 in the intracellular domain of the leptin receptor, which mediates signaling through the SH2-containing tyrosine phosphatase/extracellular signal-regulated kinase (SHP2/ERK) and signal transducer and activator of transcription 3 (STAT3) pathways, respectively, demonstrated that both were important for mucosal protection. We conclude that leptin-mediated resistance to amebiasis is via its actions on intestinal epithelium rather than hematopoietic cells or the brain, and requires leptin receptor signaling through both the STAT3 and SHP2/ERK pathways.

Zoonotic implications of the swine-transmitted protozoal infections.

Pig production is an important part of the economy in many countries. Domestic and wild pigs (Sus scrofa) are susceptible to a wide range of infectious and parasitic diseases. Some of these diseases are specifically limited to pigs while some of the other diseases are shared with other species of wildlife and domestic livestock. As the numbers and geographic distribution of wild and domestic swines continue to increase, it is certain that the number of contacts between these swines and domestic livestock will also increase, as will the probability of human exposure to the parasites of swine directly or indirectly. Here, we will discuss the protozoal infections of pigs, which have the potential to infect humans and provide reasonable risk assessment for zoonotic transmission.

Entamoeba histolytica activates host cell caspases during contact-dependent cell killing.

Entamoeba histolytica is a human intestinal parasite that causes amoebic colitis as well as liver abscesses. Host tissues are damaged through a three-step process involving adherence, contact-dependent cytolysis, and phagocytosis. These three processes all contribute to the pathogenicity of this parasite. Adherence is provided by the Gal/GalNAc adherence lectin. Host cells are lysed in a contact-dependent fashion. There is evidence that suggests that this contact-dependent killing involves the induction of the host cell's apoptotic machinery. Phagocytosis can then occur, consistent with metazoan apoptotic clearance.

Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene.

The varied organ tropisms and clinical presentations of infection by Entamoeba histolytica have stimulated interest in the role of parasite genetic diversity in virulence. We investigated genetic diversity among 54 E. histolytica isolates from Bangladesh by analyzing polymorphism in the serine-rich gene by nested PCR on DNA extracted from stool and liver aspirate pus. We detected both size and restriction site polymorphisms among the isolates within this endemic area. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded 25 distinct DNA banding patterns among the 42 stool isolates and an additional 9 distinct patterns among the 12 liver abscess isolates. Approximately half of the isolates had unique polymorphisms. Interestingly, the majority of E. histolytica from the liver had polymorphisms which were not present in intestinal isolates from the same geographic area. These data are consistent with the existence of genetic differences between E. histolytica which cause intestinal and those which cause hepatic disease. We conclude that there is genetic diversity within E. histolytica isolates from an endemic population as reflected in serine-rich E. histolytica protein gene polymorphism. The correlation of genetic differences with the pathogenic potential of E. histolytica strains and the implications of genetic diversity for the immunoprophylaxis of amebiasis require further study.

Intermediate subunit of the Gal/GalNAc lectin of Entamoeba histolytica is a member of a gene family containing multiple CXXC sequence motifs.

Killing by Entamoeba histolytica requires parasite adherence to host galactose- and N-acetyl-D-galactosamine (Gal/GalNAc)-containing cell surface receptors. A 260-kDa heterodimeric E. histolytica Gal/GalNAc lectin composed of heavy (Hgl) and light (Lgl) subunits has been previously described. Here we present the cloning and characterization of Igl, a 150-kDa intermediate subunit of the Gal/GalNAc lectin. Igl, Hgl, and Lgl colocalized on the surface membrane of trophozoites. Two unlinked copies of genes encoding Igl shared 81% amino acid sequence identity (GenBank accession no. AF337950 and AF337951). They encoded cysteine-rich proteins with amino- and carboxy-terminal hydrophobic signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored membrane proteins. The igl genes lacked carbohydrate recognition domains but were members of a large family of amebic genes containing CXXC and CXC motifs. These data indicate that Igl is part of the parasite's multimolecular Gal/GalNAc adhesin required for host interaction.

Identification and characterization of an Entamoeba histolytica upstream regulatory element 3 sequence-specific DNA-binding protein containing EF-hand motifs.

The hgl5 gene of Entamoeba histolytica is negatively regulated through the upstream regulatory element 3 (URE3) DNA motif TATTCTATT. This motif is also present and significant in the function of the E. histolytica fdx gene promoter. A yeast one-hybrid screen was used to identify an E. histolytica cDNA encoding a protein (URE3-BP) that recognized this DNA motif. Analysis of the predicted amino acid sequence demonstrated the presence of two EF-hand motifs but identified no canonical DNA binding motifs. URE3-BP, expressed in bacteria, demonstrated Ca(2+)-dependent and sequence-specific recognition of the URE3 DNA sequence as assessed by electrophoretic mobility shift assays. Antibodies raised against URE3-BP blocked the formation of the URE3 DNA-protein complex by native nuclear extracts. The URE3-BP protein was present in the E. histolytica nucleus and cytoplasm with an apparent molecular mass of 22.6 kDa. Our results represent the first use of a yeast genetic screen to identify, on the basis of function, a DNA-binding protein of an early branching eukaryote. Since the URE3 DNA can modulate gene expression in both a positive and negative manner, this protein may have more than one mechanism of interaction with transcriptional machinery. Characterization of URE3-BP should provide insight into transcription regulation and virulence control in this parasite.

Identification of two Entamoeba histolytica sequence-specific URE4 enhancer-binding proteins with homology to the RNA-binding motif RRM.

To study transcriptional regulation in the lower branching eukaryote Entamoeba histolytica, we have identified two sequence-specific DNA-binding proteins that recognize the upstream regulatory element URE4, an enhancer that regulates expression of the Gal/GalNAc lectin heavy subunit gene hgl5. A chromatographic purification of E. histolytica nuclear extracts by gel filtration, cation exchange, and sequence-specific DNA affinity chromatography led to a 700-fold increase in URE4 binding activity and the appearance of two dominant protein species with molecular masses of 28 and 18 kDa. These proteins, termed E. histolytica enhancer-binding proteins 1 and 2 (EhEBP1 and EhEBP2), were sequenced by tandem mass spectroscopy and their corresponding cDNA clones identified. Recombinant EhEBP1 and EhEBP2 were able to bind double-stranded oligonucleotides bearing the URE4 motif in a sequence-specific manner, and antibodies raised against EhEBP1 were able to interfere with the formation of URE4-protein complexes in crude nuclear extracts. Overexpression of EhEBP1 in E. histolytica trophozoites resulted in a 7-fold drop in promoter activity in transiently transfected reporter gene constructs when the URE4 motif was present, confirming its ability to specifically recognize the URE4 motif and suggesting that additional cofactors may be required for transcriptional activation by URE4. Further characterization and identification of binding partners for EhEBP1 and EhEBP2, the first proteins with demonstrated sequence-specific DNA binding activity to be identified in E. histolytica, should provide new insights into transcriptional regulation in this protozoan parasite.

Emerging and reemerging intestinal protozoa.

The intestinal protozoa have gained importance to physicians practicing medicine in the United States, Canada, and Europe during recent years as a result of increasing world travel, the globalization of the world's economy, and the growing number of chronically immunosuppressed people. During the spring of 1996, Cyclospora cayetanensis caused diarrhea in approximately 1500 people exposed to Guatemalan raspberries. This epidemic recurred in 1997, emphasizing the risks of the global economy and food supply on which we depend. In addition to importation of intestinal protozoa from the tropics, AIDS and the increasing use of organ transplants have created a new population of people at risk for chronic infection by ubiquitous protozoa previously not known to cause serious human disease. These infections include cryptosporidiosis, isosporiasis, and microsporidiosis. Finally, Entamoeba histolytica, the etiologic agent of invasive amebiasis, has only recently been recognized to be a distinct species from a nonpathogenic but indistinguishable (by light microscopy) intestinal commensal, Entamoeba dispar. The rapidly changing epidemiology of these intestinal protozoa, as well as new approaches to diagnosis and treatment of these protozoa, are discussed.